NKp46 Clusters at the Immune Synapse and Regulates NK Cell Polarization

Cell surface proteins major histocompatibility complex (MHC) class I–related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein–tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.

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Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

10.1101/266429 ◽

2018 ◽

Author(s):

David Tomaz ◽

Pedro Matos Pereira ◽

Nadia Guerra ◽

Julian Dyson ◽

Ricardo Henriques ◽

...

Keyword(s):

Nk Cell ◽

Cell Interaction ◽

Nanometer Scale ◽

Signal Integration ◽

Inhibitory Receptors ◽

Immune Synapse ◽

Cell Responses ◽

Activating Receptors ◽

Cell Target ◽

Cell Inhibition

NK cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here, we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at nanometer-scale with the activating receptor NKG2D upon immune synapse (IS) formation. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at a nanometer scale leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D consequently impairing Ly49A-mediated inhibition. Our results suggest the balance of NK cell signals, and NK cell responses, are determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse.

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AKAP350 regulates cytoskeleton remodeling and LFA-1 organization during NK cytolytic response

10.1101/2020.10.27.357970 ◽

2020 ◽

Author(s):

Alejandro P. Pariani ◽

Evangelina Almada ◽

Florencia Hidalgo ◽

Carla Borini-Etichetti ◽

Rodrigo Vena ◽

...

Keyword(s):

Golgi Apparatus ◽

Nk Cell ◽

Cell Interaction ◽

Cytolytic Activity ◽

Microtubule Dynamics ◽

Interacting Protein ◽

Immune Synapse ◽

Cytoskeleton Remodeling ◽

First Time ◽

Cytolytic Response

AbstractNatural killer (NK) cell cytotoxicity requires extensive cytoskeleton remodeling and receptors reorganization at the site of NK/target cell interaction (immune synapse, IS). How these processes are integrated remains unclear. We found that AKAP350, a scaffold protein that regulates microtubule dynamics, was required for NK cytolytic activity. Reduction of AKAP350 expression inhibited actin and LFA-1 reorganization and centrosome translocation to the IS. AKAP350KD cells showed decreased microtubule nucleation at the Golgi apparatus and reduced localization at the centrosome and at the IS of CIP4, an AKAP350 interacting protein that regulates centrosome translocation to the IS. AKAP350 delocalization from the Golgi or pharmacological disruption of microtubule dynamics or Golgi function impaired LFA-1 clustering at the IS, preserving actin remodeling. These data support a role for AKAP350 in the integration of actin and microtubule remodeling during NK activation and reveal for the first time the participation of the Golgi apparatus in NK-IS maturation.

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Ab-IL2 fusion proteins mediate NK cell immune synapse formation in epithelial ovarian cancer by polarizing CD25 to the target cell–effector cell interface

Gynecologic Oncology ◽

10.1016/j.ygyno.2010.12.103 ◽

2011 ◽

Vol 120 ◽

pp. S42

Author(s):

J. Connor ◽

I. Buhtoiarov ◽

S. Gillies ◽

J. Gubbels ◽

J. Hank ◽

...

Keyword(s):

Ovarian Cancer ◽

Epithelial Ovarian Cancer ◽

Target Cell ◽

Fusion Proteins ◽

Effector Cell ◽

Synapse Formation ◽

Nk Cell ◽

Immune Synapse ◽

Cell Interface ◽

Cell Effector

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Lenalidomide Alone and in Combination with Rituximab Enhances NK Cell Immune Synapse Formation in Chronic Lymphocytic Leukemia (CLL) Cells in Vitro through Activation of Rho and Rac1 GTPases.

Blood ◽

10.1182/blood.v114.22.3441.3441 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3441-3441 ◽

Cited By ~ 5

Author(s):

Svetlana Gaidarova ◽

JianWu Li ◽

Laura G Corral ◽

Emilia Glezer ◽

Peter H Schafer ◽

...

Keyword(s):

B Cells ◽

Nk Cells ◽

Synapse Formation ◽

Nk Cell ◽

Employment Equity ◽

Immune Synapse ◽

Immunological Synapses ◽

Immune Synapses ◽

Cd20 Expression ◽

Equity Ownership

Abstract Abstract 3441 Poster Board III-329 Background CLL is characterized by the progressive accumulation of monoclonal B lymphocytes. One theory to explain how CLL cells avoid elimination through immune surveillance mechanisms is through a defect in the ability of T-cells to form immunological synapses with antigen-presenting tumor B-cells (Ramsay et al JCI 2008). Lenalidomide is an immunomodulatory agent with clinical activity in the treatment of B-cell malignancies. Recent laboratory studies showed that lenalidomide not only stimulates T- and natural killer (NK)-cell-mediated ADCC, it also restores the T-cell-mediated ability to form immunological synapses with CLL tumor cells. Since NK cells also exert cytotoxicity through immune synapse formation, here we explore how lenalidomide affects NK-cell-mediated cytotoxicity mechanisms and whether this activity is altered in the presence of rituximab since published studies showed that lenalidomide-pretreated B-cells have a down-regulated surface CD20 expression. Further, we investigated the molecular events associated with immune synapse formation and the effect of lenalidomide. Methods Immune synapse formation was assessed in NK cells (from healthy donors PBMCs) co-cultured with either B-CLL cells derived from pts or with K562 cells (positive control). Cells were fixed and the ability to form synapses was assessed via immunohistochemisty co-staining for either F-actin and CD2, or F-actin and perforin (a cytolytic protein found in NK cells). Synapse formation was visualized by microscopy and measured via relative mean fluorescent intensity. Activity of RhoA, Rac1, Cdc42 were measured using Rho GTPases assay kits. Inhibition of lenalidomide-mediated immune synapse activity was assayed using the cell permeable Rho inhibitor C3 (0.5 mM). Flow cytometry was used to measure changes in surface CD20 and CD54 (ICAM-1) expression in B-CLL samples from 3 pts after treatment with lenalidomide. Results Lenalidomide induced the formation of immunological synapses between NK cells and primary B-CLL cells (p<.01) or the K562 cell line. Lenalidomide activated NK cells regardless of the presence of target cells, as measured by F-actin and perforin staining. RhoA and Rac1 were activated at the immunological synapse in the presence of lenalidomide. Inhibition of RhoA by the C3 inhibitor blocked F-actin localization, as well as perforin accumulation induced by lenalidomide at cell-cell contact sites, indicating inhibition of immune synapses and the associated cytolytic activity. This was also observed with Rac1 inhibition, but to a lesser degree than with RhoA inhibition. Functionality of formed synapses was confirmed by co-localization of F-actin and perforin at the synapse sites. 3 CLL pt samples treated ex vivo with lenalidomide demonstrated variable changes in CD20 expression: a 20-30% decrease in CD20 expression was observed in 2 B-CLL pt samples, whereas CD20 levels remained unchanged in the third. In the presence of rituximab, lenalidomide-induced synapse formation between NK cells and B-cells from CLL patients was further enhanced. This was accompanied by upregulation of costimulatory and adhesion molecule CD54 on B-CLL cells suggesting increased antigen presentation, which might contribute to the increased synapse formation. Conclusion Lenalidomide can directly activate NK-cell-mediated anti-tumor activity through enhanced formation of immune synapses via the regulation of Rho and Rac1 GTPases and the cytoskeleton. Despite some down-modulation of CD20 expression in lenalidomide-pretreated B-CLL cells, the immune synapse activity increases when lenalidomide is combined with rituximab suggesting that combining lenalidomide and anti-CD20 antibodies warrants exploration in the CLL clinical setting. Disclosures Gaidarova: Celgene: Employment, Equity Ownership. Li:Celgene: Employment. Corral:Celgene: Employment. Glezer:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment. Xie:Celgene: Employment. Lopez-Girona:Celgene: Employment.

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Impaired Natural Killer Cells Immune Synapse Formation in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.2663.2663 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2663-2663

Author(s):

Dongxia Xing ◽

Alan G. Ramsay ◽

William Decker ◽

Sufang Li ◽

Simon Robinson ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Immune Suppression ◽

Direct Contact ◽

Myeloid Leukemia ◽

Synapse Formation ◽

Nk Cell ◽

Immune Synapse ◽

Acute Myeloid

Abstract Abstract 2663 Poster Board II-639 Natural killer (NK) cells are an important component of the innate immune surveillance of tumor cells. Defective NK cell function has been correlated with poor prognosis in acute myeloid leukemia (AML). It is well established that NK cell-mediated cytolytic activity is significantly diminished in AML patients; the mechanisms of this hypo-function are not well understood. Identifying mechanisms of tumor-induced immune suppression of lymphocytes function will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired NK cell responses in AML and demonstrate impaired NK cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between CD56+ CD3- NK cells and autologous AML blasts. We identified a significant reduction in formation of the NK cell immune synapse (NKIS) (p<0.001) from AML patients compared healthy donors (> 70% reduction). This defect was induced by direct tumor contact since NK cell defects were induced in healthy NK cells when they were co-cultured (in direct contact) for 48 hr with allogeneic AML blasts, but not with healthy allogeneic monocytes (P < 0.01). In control transwell co-culture experiments, where the NK cells and AML blast were not in direct contact, we did not observe the induced defect. We examined the molecular nature of the AML blast induced defect by quantifying recruitment of a number of these NK cell adhesion and cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with AML blasts, healthy NK cells showed significantly reduced recruitment of integrin LFA-1, CD2, Lck, WASP, and tyrosine-phosphorylated protein to the NK-AML target interactions synapse (P < 0.001). These studies demonstrate a role for the tumor induced immune suppression of NK cells and will aid in the development of immunotherapeutic approaches targeting AML. Disclosures: No relevant conflicts of interest to declare.

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Intercellular protein transfer at the NK cell immune synapse: mechanisms and physiological significance

The FASEB Journal ◽

10.1096/fj.06-7488rev ◽

2007 ◽

Vol 21 (8) ◽

pp. 1636-1646 ◽

Cited By ~ 28

Author(s):

Pedro Roda‐Navarro ◽

Hugh T. Reyburn

Keyword(s):

Nk Cell ◽

Physiological Significance ◽

Protein Transfer ◽

Immune Synapse

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Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes

Journal of Experimental Medicine ◽

10.1084/jem.20100040 ◽

2010 ◽

Vol 207 (9) ◽

pp. 1923-1938 ◽

Cited By ~ 24

Author(s):

Aradhana Awasthi ◽

Asanga Samarakoon ◽

Haiyan Chu ◽

Rajasekaran Kamalakannan ◽

Lawrence A. Quilliam ◽

...

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Synapse Formation ◽

Nk Cell ◽

Scaffolding Protein ◽

Microtubule Organizing Center ◽

Chemokine Production ◽

Immune Synapse ◽

Cell Functions ◽

Organizing Center

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions.

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Lenalidomide in non-Hodgkin lymphoma: biological perspectives and therapeutic opportunities

Blood ◽

10.1182/blood-2014-11-567792 ◽

2015 ◽

Vol 125 (16) ◽

pp. 2471-2476 ◽

Cited By ~ 33

Author(s):

Athena Kritharis ◽

Michael Coyle ◽

Jaya Sharma ◽

Andrew M. Evens

Keyword(s):

T Cell ◽

Immune Modulation ◽

Nk Cell ◽

Predictive Biomarkers ◽

Immune Synapse ◽

Antiproliferative Effects ◽

Lymphoid Malignancies ◽

Non Hodgkin Lymphoma ◽

Lymphoma Treatment ◽

Mantle Cell

AbstractLenalidomide is an immunomodulatory drug (IMiD) with activity in lymphoid malignancies occurring primarily through immune modulation (eg, T-cell immune synapse enhancement and NK-cell/T-cell effector augmentation) and antiproliferative effects. Food and Drug Administration–approved for bortezomib-resistant, relapsed/refractory mantle-cell lymphoma, lenalidomide has demonstrated efficacy in several additional lymphoma subtypes. There are many ongoing clinical trials examining the use of lenalidomide alone or in combinatorial therapy. It will be important in these studies to delineate reliable, predictive biomarkers to optimally integrate lenalidomide into lymphoma treatment paradigms.

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